The Multiple Substrate Recognition Properties of Ribonuclease P

Abstract

Ribonuclease P is an essential enzyme that is responsible for catalyzing the 5’end maturation of transfer RNA. In Bacteria, the RNase P holoenzyme is composed of a large RNA (∼400nt) and a smaller (∼100aa) protein. RNase P RNA contains the enzyme active site and interacts with tRNA; however, the protein subunit is essential in vivo because it binds the pre-tRNA leader sequence and increases active site metal ion binding. Unlike most enzymes, RNase P processes multiple structurally distinct substrates. Although the processing kinetics of individual substrates have been examined, comparison of processing kinetics and kinetic competition between substrates has not been explored. To understand the basis for multiple substrate recognition by the holoenzyme we determined kinetic schemes forpre-tRNA608MET, a consensus pre-tRNA, and pretRNA605fmet, a non-consensus pre-tRNA, using fluorescence assays and standard discontinuous assays. Remarkably, the results show processing kinetics to be uniform with the rate limiting step being association. To obtain a complete and comprehensive understanding of how sequence and structure influence processing rates and competition between substrates, we undertook two complementary directions. First, we directly examined the competition between pre-tRNA608MET and pre-tRNA605fmet distinguishing products using different leader sequence length. Second, we designed a high throughput method to determine the effects, if any, of differences in pre-tRNA leader sequence and structure by using deep sequencing to follow the range in distribution of different substrate sequence variants in the same reaction.